- Assay conditions and analytical methods established for the
study of epoxidation of the insecticides aldrin and heptachlor, by
rat liver microsomes have been employed to study the same reaction
catalyzed by trout liver microsomes. At 37° C and pH 8.0, rainbow
trout liver microsomes, incubated with 54.8 millimicromoles of
either aldrin or heptachlor in the presence of reduced triphosphopyridine
nucleotide (NADPH) and oxygen, produced only 0.36 milli-micromole of the corresponding expoxides, dieldrin and heptachlor
epoxide. This was only 7.2% of the amount produced by similar
assays with male rat liver microsomes.
Increasing the microsomal protein and extending the incubation
time failed to increase the epoxide yield of the fish microsomes.
The same result was obtained when incubations were carried out at
2, 11, and 22° C, within a pH range of 6.0 to 8.5. Varying the substrate
level from 5 to 100 millimicromoles did not affect the activity. Dieldrin formation remained unchanged when a large excess of an
NADPH-generating system or six micromoles of either Fe(II),
Mn(II), Co(II), Mg(II) or FMN was added. Incubation of aldrin with
freshly prepared trout liver slices instead of microsomes failed to
show any improvement in epoxidation. However, no dieldrin was
detected when either heat-denatured microsomes, snake venom-treated preparation, steapsin-treated preparation or liver acetone
powder was used.
Activities of the components of the microsomal electron transport
system, which plays a vital role in epoxidation, were measured.
Results indicated their presence in significant levels in trout liver
Aldrin and heptachlor conversions by male rat liver micro-somes were enhanced two to four times by the addition of either an
acetone powder preparation, the supernatant fraction above the
78,000 X g pellet, or microsomes, of trout liver. A reaction is
said to be enhanced, when the activity of the combined system is
greater than the sum of those of the separate preparations. However,
no enhancement was observed when an inactive rat preparation,
such as a rat liver acetone powder preparation, was used in
combination with a trout fraction. The extent of enhancement in
epoxidation depended primarily on the basal activity of the rat liver
microsomes. Furthermore, the enhancing ability of the fish fractions was abolished by treating them with heat, snake venom, or steapsin.
Of the three microsomal electron transport component activities
tested, only that involved in neotretazolium (NT) reduction was
enhanced by combining rat and trout liver microsomes in the assays.
NADPH-cytochrome c and dichlorophenol indophenol (DCPIP) reductase
activities were additive in the combined systems. NT reduction
is a test reaction for component X₁ in the NADPH-dependent electron
transport chain of liver microsomes.
The parallel relationship between enhancement of NT reduction
and epoxidation and their relative positions in the established
sequence of reactions in the NADPH-dependent chain indicates that
the enhancement in epoxidation is brought about by an increase in
X₁ relative to the epoxidative enzyme(s) in the combined systems.
The evidence collected in this research leads to the conclusion that
the low epoxidative activity in trout liver microsomes is a result of
a deficiency in the epoxidative enzyme(s).